Functionalized Solid-Sphere PEG-b-PCL Nanoparticles to Target Brain Capillary Endothelial CellsIn Vitro

Nanoparticles are increasingly used to implement drug targeting strategies. In the present study, solid-sphere nanoparticles (SNPs) made of poly(ethylene glycol)-b-poly(ε-caprolactone) (PEG-b-PCL) were covalently linked to a monoclonal antibody (83-14 mAb) targeted against the human insulin receptor that is highly expressed on human brain microvascular endothelial cells. Resulting targeted SNPs were characterized using transmission electron microscopy (TEM), cryo-TEM, dynamic light scattering, and fluorescence correlation spectroscopy. The critical aggregation concentration was determined using a fluorescence approach. Interaction with a well-characterized humanin vitromodel of the blood-brain barrier (hCMEC/D3) was analysed using an array of methods (flow cytometry, confocal laser scanning microscopy, and TEM). The toxicity on hCMEC/D3 cells and in addition on the human liver cell line HepG2 was assessed using the MTT assay. SNPs with a diameter of 80 nm and a homogeneous size distribution were obtained. Successful conjugation of 83-14 mAb using a heterobifunctional linker resulted in 5-6 molecules of fluorescently labeled 83-14 mAb per SNP. Functionalized SNPs were taken up by hCMEC/D3 cells efficiently without showing a significant toxic effect on cells of the blood-brain barrier and HepG2 cells. These results indicate that functionalized PEG-b-PCL SNPs are a promising candidate to deliver drugs to the CNS.

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Transport of ultrasmall gold nanoparticles (2 nm) across the blood–brain barrier in a six-cell brain spheroid model

Scientific Reports ◽

10.1038/s41598-020-75125-2 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Viktoriya Sokolova ◽

Gehad Mekky ◽

Selina Beatrice van der Meer ◽

Michael C. Seeds ◽

Anthony J. Atala ◽

...

Keyword(s):

Gold Nanoparticles ◽

Blood Brain Barrier ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Brain Barrier ◽

Confocal Laser ◽

Hydrodynamic Diameter ◽

Core Diameter ◽

Blood Brain

Abstract The blood–brain barrier (BBB) is an efficient barrier for molecules and drugs. Multicellular 3D spheroids display reproducible BBB features and functions. The spheroids used here were composed of six brain cell types: Astrocytes, pericytes, endothelial cells, microglia cells, oligodendrocytes, and neurons. They form an in vitro BBB that regulates the transport of compounds into the spheroid. The penetration of fluorescent ultrasmall gold nanoparticles (core diameter 2 nm; hydrodynamic diameter 3–4 nm) across the BBB was studied as a function of time by confocal laser scanning microscopy, with the dissolved fluorescent dye (FAM-alkyne) as a control. The nanoparticles readily entered the interior of the spheroid, whereas the dissolved dye alone did not penetrate the BBB. We present a model that is based on a time-dependent opening of the BBB for nanoparticles, followed by a rapid diffusion into the center of the spheroid. After the spheroids underwent hypoxia (0.1% O2; 24 h), the BBB was more permeable, permitting the uptake of more nanoparticles and also of dissolved dye molecules. Together with our previous observations that such nanoparticles can easily enter cells and even the cell nucleus, these data provide evidence that ultrasmall nanoparticle can cross the blood brain barrier.

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P10.02 Combined methods of a micropump system and a chronic cranial window allows tumor observation with multi photon laser scanning microscopy under continuous treatment

Neuro-Oncology ◽

10.1093/neuonc/noab180.095 ◽

2021 ◽

Vol 23 (Supplement_2) ◽

pp. ii28-ii28

Author(s):

S Weil ◽

E Jung ◽

D Domínguez Azorín ◽

J Higgins ◽

J Reckless ◽

...

Keyword(s):

Drug Delivery ◽

Tumor Cells ◽

Blood Brain Barrier ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

New Drugs ◽

Brain Barrier ◽

Cranial Window ◽

Blood Brain ◽

The Brain

Abstract BACKGROUND Glioblastomas are notoriously therapy resistant tumors. As opposed to other tumor entities, no major advances in therapeutic success have been made in the past decades. This has been calling for a deeper biological understanding of the tumor, its growth and resistance patterns. We have been using a xenograft glioma model, where human glioblastoma cells are implanted under chronic cranial windows and studied longitudinally over many weeks and months using multi photon laser scanning microscopy (MPLSM). To test the effect of (new) drugs, a stable and direct delivery system avoiding the blood-brain-barrier has come into our interest. MATERIAL AND METHODS We implanted cranial windows and fluorescently labeled human glioblastoma stem-like cells into NMRI nude mice to follow up on the tumor development in our MPLSM model. After tumor establishment, an Alzet® micropump was implanted to directly deliver agents via a catheter system continuously over 28 days directly under the cranial window onto the brain surface. Using the MPLSM technique, the continuous delivery and infusion of drugs onto the brain and into the tumor was measured over many weeks in detail using MPLSM. RESULTS The establishment of the combined methods allowed reliable concurrent drug delivery over 28 days bypassing the blood-brain-barrier. Individual regions and tumor cells could be measured and followed up before, and after the beginning of the treatment, as well as after the end of the pump activity. Fluorescently labelled drugs were detectable in the MPLSM and its distribution into the brain parenchyma could be quantified. After the end of the micropump activity, further MPLSM measurements offer the possibility to observe long term effects of the applied drug on the tumor. CONCLUSION The combination of tumor observation in the MPSLM and concurrent continuous drug delivery is a feasible and reliable method for the investigation of (novel) anti-tumor agents, especially drugs that are not blood-brain-barrier penetrant. Morphological or even functional changes of individual tumor cells can be measured under and after treatment. These techniques can be used to test new drugs targeting the tumor, its tumor microtubes and tumor cells networks, and measure the effects longitudinally.

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Network Pharmacology-Based Approach to Revealing Biological Mechanisms of Qingkailing Injection against IschemicStroke: Focusing on Blood-Brain Barrier

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2020/2914579 ◽

2020 ◽

Vol 2020 ◽

pp. 1-16

Author(s):

Shuang Zhang ◽

Xueqian Wang ◽

Fafeng Cheng ◽

Chongyang Ma ◽

Shuning Fan ◽

...

Keyword(s):

Ischemic Stroke ◽

Tight Junction ◽

Blood Brain Barrier ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Brain Barrier ◽

Neurological Deficits ◽

Network Pharmacology ◽

Ppi Network ◽

Blood Brain

Ischemic stroke is the most common type of cerebrovascular accident worldwide. It causes long-term disability and death. Qingkailing (QKL) injection is a traditional Chinese patent medicine which has been clinically applied in the treatment of ischemic stroke for nearly thirty years. In the present study, network pharmacology combined with experimentation was used to elucidate the mechanisms of QKL. ADME screening and target prediction identified 62 active compounds and 275 targets for QKL. Topological screening of the protein-protein interaction (PPI) network was used to build a core PPI network consisting of 408 nodes and 17,830 edges. KEGG enrichment indicated that the main signaling pathway implicated in ischemic stroke involved hypoxia-inducible factor-1 (HIF-1). Experimentation showed that QKL alleviated neurological deficits, brain infraction, blood-brain barrier (BBB) leakage, and tight junction degeneration in a mouse ischemic stroke model. Two-photon laser scanning microscopy was used to evaluate BBB permeability and cerebral microvessel structure in living mice. HIF-1α, matrix metalloproteinase-9 (MMP-9), and tight junction proteins such as occludin, zonula occludins-1 (ZO-1), claudin-5, and VE-Cadherin were measured by western blotting. QKL upregulated ZO-1 and downregulated HIF-1α and MMP-9. QKL has a multiapproach, multitarget, and synergistic effect against ischemic stroke.

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Degradation of Drug Delivery Nanocarriers and Payload Release: A Review of Physical Methods for Tracing Nanocarrier Biological Fate

Pharmaceutics ◽

10.3390/pharmaceutics13060770 ◽

2021 ◽

Vol 13 (6) ◽

pp. 770

Author(s):

Patrick M. Perrigue ◽

Richard A. Murray ◽

Angelika Mielcarek ◽

Agata Henschke ◽

Sergio E. Moya

Keyword(s):

Drug Delivery ◽

Laser Scanning ◽

Single Photon ◽

Photon Emission ◽

Correlation Spectroscopy ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Emission Computed Tomography ◽

Systemic Delivery

Nanoformulations offer multiple advantages over conventional drug delivery, enhancing solubility, biocompatibility, and bioavailability of drugs. Nanocarriers can be engineered with targeting ligands for reaching specific tissue or cells, thus reducing the side effects of payloads. Following systemic delivery, nanocarriers must deliver encapsulated drugs, usually through nanocarrier degradation. A premature degradation, or the loss of the nanocarrier coating, may prevent the drug’s delivery to the targeted tissue. Despite their importance, stability and degradation of nanocarriers in biological environments are largely not studied in the literature. Here we review techniques for tracing the fate of nanocarriers, focusing on nanocarrier degradation and drug release both intracellularly and in vivo. Intracellularly, we will discuss different fluorescence techniques: confocal laser scanning microscopy, fluorescence correlation spectroscopy, lifetime imaging, flow cytometry, etc. We also consider confocal Raman microscopy as a label-free technique to trace colocalization of nanocarriers and drugs. In vivo we will consider fluorescence and nuclear imaging for tracing nanocarriers. Positron emission tomography and single-photon emission computed tomography are used for a quantitative assessment of nanocarrier and payload biodistribution. Strategies for dual radiolabelling of the nanocarriers and the payload for tracing carrier degradation, as well as the efficacy of the payload delivery in vivo, are also discussed.

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From Macro to Mesoporous ZnO Inverse Opals: Synthesis, Characterization and Tracer Diffusion Properties

Nanomaterials ◽

10.3390/nano11010196 ◽

2021 ◽

Vol 11 (1) ◽

pp. 196

Author(s):

Shravan Kousik ◽

Diane Sipp ◽

Karina Abitaev ◽

Yawen Li ◽

Thomas Sottmann ◽

...

Keyword(s):

Open Porosity ◽

Laser Scanning ◽

High Surface ◽

Stop Band ◽

Correlation Spectroscopy ◽

Laser Scanning Microscopy ◽

Added Value ◽

Confocal Laser ◽

Inverse Opals ◽

Mesoporous Zno

Oxide inverse opals (IOs) with their high surface area and open porosity are promising candidates for catalyst support applications. Supports with confined mesoporous domains are of added value to heterogeneous catalysis. However, the fabrication of IOs with mesoporous or sub-macroporous voids (<100 nm) continues to be a challenge, and the diffusion of tracers in quasi-mesoporous IOs is yet to be adequately studied. In order to address these two problems, we synthesized ZnO IOs films with tunable pore sizes using chemical bath deposition and template-based approach. By decreasing the size of polystyrene (PS) template particles towards the mesoporous range, ZnO IOs with 50 nm-sized pores and open porosity were synthesized. The effect of the template-removal method on the pore geometry (spherical vs. gyroidal) was studied. The infiltration depth in the template was determined, and the factors influencing infiltration were assessed. The crystallinity and photonic stop-band of the IOs were studied using X-Ray diffraction and UV-Vis, respectively. The infiltration of tracer molecules (Alexa Fluor 488) in multilayered quasi-mesoporous ZnO IOs was confirmed via confocal laser scanning microscopy, while fluorescence correlation spectroscopy analysis revealed two distinct diffusion times in IOs assigned to diffusion through the pores (fast) and adsorption on the pore walls (slow).

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Studying the Neurovascular Unit: An Improved Blood–Brain Barrier Model

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2009.103 ◽

2009 ◽

Vol 29 (12) ◽

pp. 1879-1884 ◽

Cited By ~ 19

Author(s):

Christoph M Zehendner ◽

Heiko J Luhmann ◽

Christoph RW Kuhlmann

Keyword(s):

Blood Brain Barrier ◽

Laser Scanning ◽

Neurovascular Unit ◽

Cell Types ◽

Laser Scanning Confocal Microscopy ◽

Brain Barrier ◽

Scanning Confocal Microscopy ◽

Blood Brain

The blood–brain barrier (BBB) closely interacts with the neuronal parenchyma in vivo. To replicate this interdependence in vitro, we established a murine coculture model composed of brain endothelial cell (BEC) monolayers with cortical organotypic slice cultures. The morphology of cell types, expression of tight junctions, formation of reactive oxygen species, caspase-3 activity in BECs, and alterations of electrical resistance under physiologic and pathophysiological conditions were investigated. This new BBB model allows the application of techniques such as laser scanning confocal microscopy, immunohistochemistry, fluorescent live cell imaging, and electrical cell substrate impedance sensing in real time for studying the dynamics of BBB function under defined conditions.

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Confocal-based fluorescence fluctuation spectroscopy with a SPAD array detector

Light Science & Applications ◽

10.1038/s41377-021-00475-z ◽

2021 ◽

Vol 10 (1) ◽

Cited By ~ 1

Author(s):

Eli Slenders ◽

Marco Castello ◽

Mauro Buttafava ◽

Federica Villa ◽

Alberto Tosi ◽

...

Keyword(s):

Laser Scanning ◽

Single Photon ◽

Function Analysis ◽

Pair Correlation ◽

Correlation Spectroscopy ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Array Detector ◽

Fluorescence Lifetime Imaging ◽

Fluorescence Fluctuation Spectroscopy

AbstractThe combination of confocal laser-scanning microscopy (CLSM) and fluorescence fluctuation spectroscopy (FFS) is a powerful tool in studying fast, sub-resolution biomolecular processes in living cells. A detector array can further enhance CLSM-based FFS techniques, as it allows the simultaneous acquisition of several samples–essentially images—of the CLSM detection volume. However, the detector arrays that have previously been proposed for this purpose require tedious data corrections and preclude the combination of FFS with single-photon techniques, such as fluorescence lifetime imaging. Here, we solve these limitations by integrating a novel single-photon-avalanche-diode (SPAD) array detector in a CLSM system. We validate this new implementation on a series of FFS analyses: spot-variation fluorescence correlation spectroscopy, pair-correlation function analysis, and image-derived mean squared displacement analysis. We predict that the unique combination of spatial and temporal information provided by our detector will make the proposed architecture the method of choice for CLSM-based FFS.

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Determination of Dynamics of Plant Plasma Membrane Proteins with Fluorescence Recovery and Raster Image Correlation Spectroscopy

Microscopy and Microanalysis ◽

10.1017/s1431927616000568 ◽

2016 ◽

Vol 22 (2) ◽

pp. 290-299 ◽

Cited By ~ 8

Author(s):

Martina Laňková ◽

Jana Humpolíčková ◽

Stanislav Vosolsobě ◽

Zdeněk Cit ◽

Jozef Lacek ◽

...

Keyword(s):

Plasma Membrane ◽

Laser Scanning ◽

Correlation Spectroscopy ◽

Laser Scanning Microscopy ◽

Image Correlation ◽

Fluorescence Recovery ◽

Confocal Laser ◽

Raster Image ◽

Image Correlation Spectroscopy ◽

Plant Plasma Membrane

AbstractA number of fluorescence microscopy techniques are described to study dynamics of fluorescently labeled proteins, lipids, nucleic acids, and whole organelles. However, for studies of plant plasma membrane (PM) proteins, the number of these techniques is still limited because of the high complexity of processes that determine the dynamics of PM proteins and the existence of cell wall. Here, we report on the usage of raster image correlation spectroscopy (RICS) for studies of integral PM proteins in suspension-cultured tobacco cells and show its potential in comparison with the more widely used fluorescence recovery after photobleaching method. For RICS, a set of microscopy images is obtained by single-photon confocal laser scanning microscopy (CLSM). Fluorescence fluctuations are subsequently correlated between individual pixels and the information on protein mobility are extracted using a model that considers processes generating the fluctuations such as diffusion and chemical binding reactions. As we show here using an example of two integral PM transporters of the plant hormone auxin, RICS uncovered their distinct short-distance lateral mobility within the PM that is dependent on cytoskeleton and sterol composition of the PM. RICS, which is routinely accessible on modern CLSM instruments, thus represents a valuable approach for studies of dynamics of PM proteins in plants.

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Twin-Chain Polymer Networks Loaded With Nanostructured Fluids for the Selective Removal of A Non-Original Varnish From A 20th Century Oil Painting At The Peggy Guggenheim Collection, Venice

10.21203/rs.3.rs-27616/v1 ◽

2020 ◽

Author(s):

Luciano Pensabene Buemi ◽

Maria Laura Petruzzellis ◽

David Chelazzi ◽

Michele Baglioni ◽

Rosangela Mastrangelo ◽

...

Keyword(s):

Laser Scanning ◽

Polymer Network ◽

Polymer Networks ◽

Mechanical Action ◽

Correlation Spectroscopy ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Oil Painting ◽

Chain Polymer ◽

Osmotic Balance

Abstract This paper reports on the evaluation of a polyvinyl alcohol (PVA) “twin-chain” polymer network (TC-PN) combined with an oil-in-water nanostructured fluid (NSF) for the removal of a polyvinyl acetate (PVAc) varnish. Small Angle X-ray Scattering, Confocal Laser Scanning Microscopy, and Fluorescence Correlation Spectroscopy showed that the structure of the gel and the NSF are only minimally altered by loading the fluid into the gel. The NSF is partially free to diffuse through the network, but also interacts with the gel walls. During the cleaning, the dynamics of the fluid at the gel-substrate interface are controlled by the osmotic balance taking place among the interconnected pores. These features grant effective and controlled cleaning performances. The case study identified for this research is Pablo Picasso’s The Studio (L’Atelier, 1928), one of the masterpieces in the Peggy Guggenheim Collection, Venice (PGC). In 1969 the oil painting, originally unprotected, was wax-lined and then varnished using a PVAc varnish. Over the years, the white shades of the painting have been compromised by the yellowing of the varnish and soiling of deposits. On painting mock-ups, the NSF-loaded hydrogels allowed the swelling and softening of PVAc varnish and wax layers, which were then removed with gentle mechanical action. Effective varnish and wax removal at the micron scale, and the absence of residues from the cleaning system (gel and NSF), were confirmed by Fourier Transform Infrared Spectroscopy (FTIR) 2D imaging. The effective and safe removal of the aged PVAc varnish and wax layer from the surface of the painting was then carried out using the same cleaning protocol successfully tested on the mock-ups, setting the NSF-loaded PVA TC-PNs as robust and reliable tools for the cleaning of sensitive works of art.

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QUANTITATIVE CONFOCAL LASER SCANNING MICROSCOPY

Image Analysis & Stereology ◽

10.5566/ias.v25.p111-120 ◽

2011 ◽

Vol 25 (3) ◽

pp. 111 ◽

Cited By ~ 1

Author(s):

Merete Krog Raarup ◽

Jens Randel Nyengaard

Keyword(s):

Confocal Laser Scanning Microscopy ◽

Laser Scanning ◽

3D Structure ◽

Correlation Spectroscopy ◽

Laser Scanning Microscopy ◽

Confocal Laser Scanning ◽

Scanning Microscopy ◽

Confocal Laser ◽

Two Photon ◽

Photon Excitation

This paper discusses recent advances in confocal laser scanning microscopy (CLSM) for imaging of 3D structure as well as quantitative characterization of biomolecular interactions and diffusion behaviour by means of one- and two-photon excitation. The use of CLSM for improved stereological length estimation in thick (up to 0.5 mm) tissue is proposed. The techniques of FRET (Fluorescence Resonance Energy Transfer), FLIM (Fluorescence Lifetime Imaging Microscopy), FCS (Fluorescence Correlation Spectroscopy) and FRAP (Fluorescence Recovery After Photobleaching) are introduced and their applicability for quantitative imaging of biomolecular (co-)localization and trafficking in live cells described. The advantage of two-photon versus one-photon excitation in relation to these techniques is discussed.

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